Pyrethrin which is a secondary metabolite contained in pyrethrum exhibits excellent insecticidal activity against insects as well as being an ideal feature as an insecticidal constituent where toxicity against mammals is low, and is widely used for mosquito coils, and insecticide sprays and powders. Recently, demand for pyrethrin has decreased because of the remarkable development of synthetic pyrethroid. However, pyrethrin still has a high utility value as a plant-derived, and environmentally friendly material for insecticides, and further investigation has continued to a point where pyrethrin can be obtained inexpensively and effectively. In particular, the existence value of the above pyrethrin, a secondary metabolite, has been emphasized again, because of increasing oil prices, which is a raw material of synthetic pyrethroid, and the like.
Pyrethrin is mainly extracted from the flower part of pyrethrum. However, the growth duration of pyrethrum until flowering is very long, over three years. It is considered that selection and breeding of high-producing strains of pyrethrum and promotion of pyrethrin biosynthesis in plant cells of the same or different species have a beneficial effect as a means for increasing the efficiency of pyrethrin production.
Pyrethrin has an ester-bonded structure between chrysanthemic acid that is a monoterpene carboxylic acid and rethrolones (alcohols), which is a metabolite of fatty acid oxidation (FIG. 3). It is known that in biosynthesis of pyrethrin, chrysanthemic acid and rethrolones are biosynthesized in different metabolic pathways and an ester binding is eventually formed therebetween.
Methods for increasing efficiency of the above-described biosynthesis of pyrethrin include use of genes involved in the biosynthesis. In order to implement biosynthesis of pyrethrin, isolation and identification of the relevant gene is crucial.
Meanwhile, various ester compounds produced by plant cells are biosynthesized by catalysis of acyltransferase from CoA thioester of carboxylic acid (acyl-CoA, RCO-S-CoA) and alcohol (R′—OH) as substrates (FIG. 4), and these biosyntheses are described, for example, in Non-Patent Document 1 and 2.
As an example of such acyltransferase in the pyrethrin biosynthesis, existence of chrysanthemoyl/pyrethroyl transferase (pyrethrin biosynthetic enzyme) which uses (1R)-trans-chrysanthemoyl-CoA and (S)-pyrethrolone as substrates have been predicted, however, there has been no isolated and specific composition based on an amino acid sequence, and naturally a gene encoding the protein based on such an amino acid sequence is not particularly sought.
Meanwhile, Japanese Patent Application Publication No. H9-504684 discloses an amino acid sequence of chrysanthemyl diphosphate synthase, an enzyme that can catalyze synthesis of chrysanthemyl diphosphate, which is adopted as a raw material for chemical synthesis of pyrethrin, and a sequence of a gene coding a protein based on such an amino acid sequence. However, there has been neither disclosure nor suggestion about the gene encoding the enzyme per se, which can catalyze the above pyrethrin biosynthesis, and the gene coding protein based on such an amino acid sequence. As obvious from the situation in the conventional art, elucidation of the gene encoding the above enzyme protein through identification of the enzyme involved in the pyrethrin biosynthesis, and effective biosynthesis of pyrethrin based on knowledge of genetic engineering have not been achieved.
[Patent Document 1] Japanese patent application publication No. H9-504684
[Non-Patent Document 1] R. Kalscheuer and A. Steinbuchel, A novel bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J. Biol. Chem. 278:8075-8082 (2003)
[Non-Patent Document 2] J. Luo et al., Convergent evolution in the BAHD family of acyl transferases: identification and characterization of anthocyanin acyl transferases from Arabidopsis thaliana. Plant J. 50:678-695 (2007)